LRET investigations of conformational changes in the ligand binding domain of a functional AMPA receptor

Abstract

The structural investigations using the soluble ligand binding domain of the AMPA subtype of the glutamate receptor have provided invaluable insight into the mechanistic pathway by which agonist binding to this extracellular domain mediates the formation of cation-selective channels in this protein. These structures, however, are in the absence of the transmembrane segments, the primary functional component of the protein. Here, we have used a modified luminescence resonance energy transfer based method to obtain distance changes due to agonist binding in the ligand binding domain in the presence of the transmembrane segments. These distance changes show that the cleft closure conformational change observed in the isolated ligand binding domain upon binding agonist is conserved in the receptor with the channel segments, thus establishing that the isolated ligand binding domain is a good model of the domain in the receptor containing the transmembrane segments.

Figure 1

A schematic representation of the experiments performed with the AMPA receptors expressed in oocytes. The modified GluR4 homomeric AMPA receptor had a histidine tag followed by a glutamine residue introduced at the N-terminus (histag-ΔN*-AMPA-S653C receptor). LRET was measured between the site C653 and the histidine tag before and after TAGZyme digestion, with glutamine acting as the stop site for the enzyme.

Figure 3

Functional characterization of the histag-ΔN*-AMPA-S653C receptors. (A) Radioactive ligand binding showing [³H]-AMPA binding to membrane preparations of oocytes expressing histag-ΔN*-AMPA-S653C. (B) Two electrode voltage clamp experiments performed with oocytes expressing histag-ΔN*-AMPA-S653C receptors. Representative currents recorded with 1 mM glutamate in the presence of cyclothiazide and 1 mM kainate in the presence of cyclothiazide. (C) Dose response curve showing the dependence of the maximum current as a function of glutamate concentration for the histag-ΔN*-AMPA-S653C receptors. All currents were recorded in the presence of cyclothiazide and normalized to currents mediated by 10 mM glutamate.

Figure 4

(I) LRET lifetimes as measured by the sensitized emission of the acceptor at 575 nm before and after TAGZyme digestion in the apo state for the modified AMPA receptor. (II) The difference between the lifetimes obtained before and after digestion with TAGZyme in the apo, glutamate-bound, and kainate-bound states.